Delivery of pDNA to the Lung by Lipopolyplexes Using N-Lauroylsarcosine and Effect on the Pulmonary Fibrosis

In a previous study, we constructed a lung-targeting lipopolyplex containing polyethyleneimine (PEI), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), and N-lauroylsarcosine (LS). The lipopolyplex exhibited an extremely high gene expression in the lung after intravenous administration. Here, we optimized the lipopolyplex and used it to deliver a TGF-β1 shRNA to treat refractory pulmonary fibrosis. We constructed several lipopolyplexes with pDNA, various cationic polymers, cationic lipids, and LS to select the most effective formulation. Then, the pDNA encoding shRNA against mouse TGF-β1 was encapsulated in the lipopolyplex and injected into mice with bleomycin-induced pulmonary fibrosis. After optimizing the lipopolyplex, dendrigraft poly-L-lysine (DGL) and DOTMA were selected as the appropriate cationic polymer and lipid, respectively. The lipopolyplex was constructed with a pDNA, DGL, DOTMA, and LS charge ratio of 1:2:2:4 showed the highest gene expression. After intravenous administration of the lipopolyplex, the highest gene expression was observed in the lung. In the in vitro experiment, the lipopolyplex delivered pDNA into the cells via endocytosis. As a result, the lipopolyplex containing pDNA encoding TGF-β1 shRNA significantly decreased hydroxyproline in the pulmonary fibrosis model mice. We have successfully inhibited pulmonary fibrosis using a novel lung-targeting lipopolyplex.

Novel Lung Targeting Cell Penetrating Peptides as Vectors for Delivery of Therapeutics

Cell penetrating peptides are unique, 5-30 amino acid long peptides that are able to breach cell membrane barriers and carry cargoes intracellularly in a functional form. Our prior work identified a synthetic, non-naturally occurring 12-amino acid long peptide that we termed cardiac targeting peptide (CTP: APWHLSSQYSRT) due to its ability to transduce cardiomyocytes in vivo. Studies looking into its mechanism of transduction identified two lung targeting peptides (LTPs), S7A (APWHLSAQYSRT) and R11A (APWHLSSQYSAT). These peptides robustly transduced human bronchial epithelial cell lines in vitro and mouse lung tissue in vivo. This uptake occurred independently of clathrin mediated endocytosis. Biodistribution studies of R11A showed peak uptake at 15 minutes with uptake in liver but not kidneys, indicating primarily a hepatobiliary mode of excretion. Cyclic version of both peptides was ~100-fold more efficient in permeating cells than their linear counterparts. As proof of principle, we conjugated anti-spike and anti-envelope SARS-CoV-2 siRNAs to cyclized R11A and demonstrate anti-viral efficacy in vitro. Our work presented here identifies two novel lung-specific cell penetrating peptides that could potentially deliver myriad therapeutic cargoes to lung tissue.

Effect of Sizes on the Lung-Targeting and Biocompatibility of Intravenously Administered Biodegradable PLGA Microspheres

Background: In the treatment of lung diseases, drug showed low bioavailability and efficacy by conventional administration methods. Passive lung-targeting microspheres provide a method to deliver drugs from the vascular side, there is still a paucity of systematic studies on the biocompatibility of biodegradable-polymer microspheres. Results: We used poly (lactic acid-glycolic acid) (PLGA) microspheres with different particle sizes (3, 10, 25, and 40 mm) as a model. The optimal lung-targeting particle size of the PLGA microspheres is 10 mm and the corresponding range of maximum tolerated dose is 125-150 mg/kg. We hypothesized that the decrease of blood oxygen saturation under treatment of microspheres was caused by pulmonary embolism. We found varying degrees of blood circulation loss of lungs by micro computed tomography test, which indicated severe pulmonary embolism subsequent to intravenous injection of microspheres with a particle size >25 mm. Furthermore, we found lung injury and microspheres leaked from the blood vessels into the alveoli by H&E staining. This process was likely induced by increased secretion of matrix metalloproteinases (MMPs), which destroyed the alveolar-capillary barrier and trigger an inflammatory cascade. Finally, we found that optimal particle size and dose conditions did not affect normal physiological activities after the microspheres degraded. The upregulation of vascular endothelial growth factor (VEGF) and platelet endothelial cell adhesion molecule-1 (PECAM-1 or CD31) induced vessel recanalization and reestablishment, which promoted the progress of lung repair. Conclusions: Collectively, our results highlight the importance of accurately designing the optimal size and dose of microspheres for passive lung-targeting delivery.